Interstitial fluid (ISF) flow has many functions including the maintenance of ionic balances, flushing of wastes and providing a route for migration of both cell signals and cells. Recently, the use of multiphoton microscopy, which enables in vivo studies with cellular resolution, has resulted in novel findings, especially in the brain, about dynamics and anatomy involved in ISF flow. Notably, ISF flow may be critical in dealing with protein accumulation in Alzheimer's disease and is regulated by sleep. Although much progress has been made, there remains controversy about some of the fundamentals regarding ISF flow. Much of this may be due to complications in the experimental methods. Studies to date require the injection of tracers which can be imaged by multiphoton microscopy or other imaging methods such as MRI. However, the process of injection of the tracers may itself affect the flow due to the delicate balance of pressures within the brain. In addition, injected tracers do not mimic the origin of proteins, wastes and cytokines made by the brain. Studies are limited to superficial sites accessible by current generation imaging technologies. This proposal generates optical and biological tools that address these short comings using in vivo multiphoton microscopy. First, in a new way to generate tracers in situ, cells within the tissue of interests will be transduced so that they secrete fluorescent proteins into the extracellular space. This will be used to resolve existing controversies about the route of ISF flow within the brain. Second, the newly discovered brain lymphatics are thought to link to the peri or paravacular spaces that serve as conduits for ISF. This work will use the new secrete tracers to answer whether and how these lymphatic channels link to the these spaces. This fluid flow may be altered in different conditions, so this will be studied in normal function mimicking sleep and waking, as well as with a stroke model. Third, three-photon microscopy now enables much deeper imaging than traditional two-photon microscopy. This enables imaging of anatomy previously not accessible. This work will study ISF flow in the hippocampus, a critical brain structure in memory and cognition, that seems to be particularly vulnerable to disruptions to ISF. This work will establish novel tools that can enable new experiments to address ISF in many systems.